Our approach to dissecting the processes of endocytosis, glycoprotein biosynthesis and sorting is through isolation and analysis of mutants. By hybridization we have shown that the majority of CHO cell endocytosis mutants isolated by ourselves and others fall into two genetic complementation groups, End1 and End2. Phenotypes of these two classes of mutants are essentially identical, both defects resulting in decreased acidification of early endosomes. Comparing mutant and parental cells by two-dimensional electrophoresis we have identified a membrane-associated protein that may represent End2. In the End1 and 2 CHO cell mutants a single genetic defect results in alterations of both endosomal and Golgi activities, specifically of the late Golgi-trans Golgi reticulum region. An LTk- cell mutant, altered in endocytic activity but apparently normal with respect to endosomal acidification, appears to transport viral envelope glycoproteins at a decrease rate once those proteins have entered the early regions of the Golgi. The lipid-linked oligosaccharide that is transferred en bloc to protein in N-linked glycosylation is thought to be assembled in stages, with the Man5GlcNac2 built on the cytoplasmic face of the ER using GDP-mannose, then elongated on the luminal face using ManP-dolichol. We have isolated a mutant whose phenotype is consistent with a defect in the translocation of the lipid-linked Man5 intermediate. Development of an in vitro assay system for translocation is in progress.